JPH0672845B2 - Analysis method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantitatively analyzing a specific component in a fluid by utilizing an optical change such as colorimetric analysis. In particular, it relates to reducing the time required for such analysis.

[Disadvantages of Prior Art] In order to detect a specific substance existing in a liquid, a color developed by a reaction between a substance to be detected and an appropriate reagent,
A method utilizing an optical change such as discoloration is a very general method and is called colorimetric analysis. In the wet method, a reagent that reacts with a test substance and causes an optical change such as color development is
It is added to the sample solution. On the other hand, in the recently developed dry method, a liquid sample is dropped on an analytical element containing a reagent, and the color development or the like caused by the reaction between the test substance and the reagent is measured. Examples of such dry analytical elements include those disclosed in Japanese Examined Patent Publication No. 53-21677 and Japanese Unexamined Patent Publication No. 55-164356.

When a reagent is added to the sample solution or the sample solution is spotted on the dry analytical element, the reagent and the test component react with each other to form a dye or the like. For example, when blood or plasma is spotted on the dry analytical element described in JP-A-55-164356, the reagent in the analytical element reacts with glucose in the sample to form a dye, for example, a red dye. Since the optical density of the dry analytical element corresponds to the amount of the produced dye, the reflection optical density is measured after a certain period of time, and the glucose concentration is obtained from the calibration curve previously obtained,
Convert to so-called blood sugar level. In the wet method, the permeation concentration of the liquid is measured.

Conventionally, except for the case of the analysis by the reaction rate method in which the activity of an enzyme or the like is measured using the reaction rate as the speed, the optical density measurement was only performed after a fixed time from the spotting of the sample solution or the addition of the reagent. When the concentration of the test component (for example, blood glucose level) is relatively low, the reaction has already been completed, so it is not necessary to wait until this time, which is uneconomical in time. This is all the more true with the dry analysis method in which the time required for the analysis operation is shortened. However, when the concentration of the test component is high, when the reaction time is short, the slope of the calibration curve is small, the coefficient of variation of the analytical value is large, and sufficient analytical accuracy is often not obtained.

[Technical problem to be solved] An object of the present invention is to reduce the time required for analysis to the minimum necessary without impairing analysis accuracy in a method of measuring the concentration of a specific substance in a liquid by colorimetric analysis or the like. And

[Means for Solving Problems] In the present invention, (1) the change in optical density is tracked by measuring the optical density a plurality of times at appropriate time intervals after the start of the reaction. From the value, it is judged whether or not the reaction should be continued. (3) If it is judged that it is not necessary to continue the reaction because of the analytical accuracy, select the calibration curve corresponding to the reaction time, and (4) When it is determined that the reaction needs to be continued, the reaction is further continued, and (5) the analysis is completed in the minimum required reaction time from the viewpoint of accuracy.

INDUSTRIAL APPLICABILITY The present invention can be applied to colorimetric analysis based on various color development reactions, particularly colorimetric analysis using an integrated dry analytical element. For example, it can be applied to the colorimetric analysis based on the color development reaction as listed below.

(1) Various color-developing reactions for detecting hydrogen peroxide produced directly or indirectly by a reaction with a test substance (also called an analyte). For example, in the presence of peroxidase and other peroxide catalytically active substances, phenazones (1-phenyl-2,3-dimethyl-4-aminopyrazolin-5-one, 1- (2,4,6-
Trichlorophenyl-2,3-dimethyl 4-4-aminopyrazolin-5-one etc.) and phenols (phenol,
α-naphthol, 1,7-dihydroxynaphthalene, etc.), a color change reaction (eg, benzidine, O-toluidine, O-dianisidine, tetramethylbenzidine) in the presence of peroxidase, etc. ,
U.S. Pat. No. 2,981,606), a dye forming reaction from a leuco dye having an imidazole nucleus, such as 2,4,5-triarylimidazole and 2,4-diaryl-5-alkylimidazole.

(2) Formazan dye formation reaction from a tetrazolium salt via an electron transfer agent coupled to a redox reaction between NAD and NADH or NADP and NADPH. For example, JP-A-59-8809
Pigmentation reaction from the tetrazolium salt described in No. 7.

(3) Azobilirubin formation reaction by binding of an aromatic diazonium salt and bilirubin. For example, USP 2,854,317,
See 3,880,588 and JP-A-59-145965.

(4) Valylmandelic acid and diazonium salts such as p-
Formation reaction of azo dyes with nitrobenzenediazonium.

(5) Pigment formation reaction from creatinine and picrate (so-called Jaffe method).

(6) Dye dissociation from a self-developing substrate under enzymatic activity.
For example, a hydrolysis reaction for producing para-nitrophenol from a p-nitrophenol-substituted oligosaccharide described in USP 4,233,403, a hydrolysis reaction for producing para-nitrophenol from γ-glutamyl-para-nitroanilide, para-nitrophenol phosphate, etc. Decomposition reaction.

(7) Naphthylamines such as N- (1-
Reaction of naphthyl) -N'-diethylethylenediamine with o-phthalaldehyde and urea, for example JP-A-55-6903.
8 and the reaction described in JP-A-58-117457.

(8) Metal ions such as calcium ions and chelating agents such as 3,3'-bis [{di (carboxymethyl)
Chelate dye formation reaction with amino} methyl] -o-cresolphthalein.

(9) Discoloration of acid-base indicator due to pH. For example, discoloration of phenol sulphophthalein, bromcresol green, bromcresol purple, etc.

(10) Discoloration of acid-base indicator due to protein. For example, discoloration by albumin such as bromcresol green, bromcresol purple, and tetrabromophenol blue, which are used for analysis of ammonia, urea and the like.

(11) Coloring of Biuret's reagent in alkaline environment used for quantification of total protein.

The present invention can be applied not only to the formation or discoloration of dyes (change in absorption wavelength) but also to the measurement of fading of dyes or reduction of colored substances. For example, a glucose assay that measures a decrease in NADH or NADPH and a glucose assay that measures a decrease in ferricyanide.

The present invention can be applied to the case of measuring fluorescence generated by excitation of short-wave electromagnetic waves such as ultraviolet rays and radiation. The present invention can also be applied to the case of measuring luminescence such as chemiluminescence and bioluminescence. For example, luminol emission due to the action of hydrogen peroxide.

[Effects of the Invention] In the present invention, in the method of measuring the concentration of a specific substance in a liquid by colorimetric analysis or the like, the time required for the analysis can be shortened to the necessary minimum without impairing the analysis accuracy.

Compared with the conventional end point method analysis in which the optical density measurement is performed after a certain period of time from the spotting of the sample solution or the addition of the reagent, in the present invention, when the concentration of the test component (for example, blood glucose level) is relatively low, the reaction Since the analysis is completed in a relatively short time, the analysis can be completed without waiting any longer, and the analysis time can be saved. The advantage can be utilized especially in the analytical method in which the time required for the analytical operation is shortened, such as the dry analytical method.

On the other hand, when the concentration of the test component is high, when the reaction time is short, the slope of the calibration curve is small, the coefficient of variation of the analytical value becomes large, and sufficient analytical accuracy may not be obtained. In some cases, by taking the necessary reaction time, the analysis accuracy can be kept high. This is important because the above tendency is often seen in the analytical method using the dry analytical element.

In the present invention, not only different shortening of the analysis time, but depending on the concentration of the test component in the sample solution, the minimum reaction time required on the analysis accuracy is selected, so that the required time can be reduced without impairing the analysis accuracy. Reasonable shortening can be achieved.

[Example] <Preparation of chemical analysis slide> A glucose dry chemical analysis slide was prepared as follows.

On a 180-micron-thick polyethylene terephthalate smooth film undercoated with gelatin, a reagent layer having the following composition was applied as a coating solution to a dry thickness of 15 microns and dried.

Gelatin 20 g Peroxidase 2500 IU Glucose oxidase 1000 IU 1,7-Dihydroxynaphthalene 0.5 g 4-Aminoantipyrine 0.5 g Polyoxyethylene nonylphenol 0.2 g Water 200 ml On top of this, apply a light shielding layer coating solution of the following composition to a dry thickness of 7 microns. Then, it was applied and dried.

Gelatin 10 g Titanium dioxide 100 g Water 500 ml An adhesive layer having the following composition was coated on the light shielding layer so that the dry film thickness was 2 μm, and dried.

Gelatin 4 g Polyoxyethylene nonylphenol 0.1 g Water 200 ml The adhesive layer was moistened with water at a rate of 30 g / m 2, and then cotton broad cloth was lightly pressed and dried.

Glucose analysis film prepared as described above, 15
It was cut into x15mm and stored in a 24x28mm plastic mount.

<Colorimetric analysis operation> The glucose analysis slide prepared as described above was loaded into the slide insertion position 2 of the analyzer shown in FIG. 1, and 10 μl of the sample solution was spotted with a micropipette, and immediately thereafter, the slide feed lever was used. 4 is pushed to the photometric position 3 and the slide is fed into the incubator 7.

Two minutes after the slide was fed by a timer provided in the analyzer, the optical density measurement head (probe) for measuring the reflection optical density of the chemical analysis film in the slide from the support side was passed through a photometric window provided below the incubator. ) 12 light sources, a photometric unit 14, and a reference light photometric unit 15. A filter with a maximum transmission wavelength of 500 nm was inserted in the optical path as the light source.

A flow chart showing a typical process in the present invention,
The case of this embodiment is shown in FIG.

When the value of optical density measured 2 minutes after the spotting was less than 0.400, the reaction was stopped and the slide was discharged from the incubator to the tray 23, while using the prepared calibration curve at the reaction time of 2 minutes, glucose was used. Calculate the concentration.
The calibration curve stored in the storage device in the arithmetic unit is selected by a command signal from the arithmetic control unit, input to the arithmetic unit of the arithmetic unit, and used for the arithmetic.

When the optical density after 2 minutes is 0.400 or more, the reaction on the slide is continued in the incubator until after 3 minutes, and the optical density is measured.

When the optical density after 3 minutes is less than 0.600, the reaction is stopped and the slide is ejected from the incubator, while the glucose concentration is calculated using the prepared calibration curve at the reaction time of 3 minutes. When the optical density after 3 minutes is 0.600 or more, the reaction on the slide is continued in the incubator until after 4 minutes, and the optical density is measured after 4 minutes.

When the optical density after 4 minutes is less than 0.700, the reaction is stopped and the slide is ejected from the incubator, while the glucose concentration is calculated using the prepared calibration curve at the reaction time of 4 minutes. When the optical density after 4 minutes is 0.700 or more, the reaction on the slide is continued in the incubator until after 5 minutes, and the optical density is measured after 5 minutes.

When the optical density after 5 minutes is less than 0.850, the reaction is terminated and the slide is ejected from the incubator, while the glucose concentration is calculated using the prepared calibration curve at the reaction time of 5 minutes. When the optical density after 5 minutes is 0.850 or more, the reaction on the slide is continued in the incubator until 6 minutes later, and the optical density is measured after 6 minutes. The slide after the measurement is ejected.

A third flowchart of the above logical processing in the present embodiment is shown.
As shown in the figure.

<Preparation of calibration curve> A calibration curve for each reaction time was prepared as follows prior to analysis.

On the glucose analysis slide above, 0,50,100,150,200,2
Control serum containing 50,300,350,400,450,500,550,600 mg / dl glucose respectively (commercial control serum and the required amount of glucose added) is spotted using a micropipette, and after 1,2,3,4,5,6 minutes The reflection optical density of was measured. The relationship between the reaction time and the reflection optical density at each glucose concentration was as shown in FIG. Based on this graph, a calibration curve (glucose content vs. optical density) at reaction times of 2, 3, 4, 5 and 6 minutes was obtained. The calibration curve for each reaction time was as shown in FIG.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of an analyzer used in Examples and a schematic view of an electric circuit. FIG. 2 is a typical example of a process flow chart, and FIG. 3 is a logic process flow chart of the embodiment. FIG. 4 is a graph showing the relationship between reaction time and reflection optical density in the examples, and FIG. 5 is a graph showing the calibration curve in the examples. Meanings of symbols in FIGS. 1 and 2 are as follows. 1 …… Indicates the slide discharge position of the slide insertion lever 2 …… Indicates the liquid spotting position of the slide insertion lever 3 …… Indicates the metering position of the slide insertion lever 4 Slide insertion lever 5 …… Slide insertion lever ( (Plan view) 6 …… Slide feed unit 7 …… Upper heat insulation member 8 …… Chemical analysis slide, 16 …… Lamp 9 …… Blackboard, 17 …… Amplifier 10 …… White plate, 18 …… Amplifier 11 …… Lower heat insulation member , 19 ...... Switcher 12 ...... Metering head, 20 ...... A / D converter 13 ...... Lens, 21 ...... Computer 14 ...... Light receiving element, 22 ...... Base 15 ...... Light receiving element (reference light) 23 ... … Slide ejection tray

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Claims (1)

[Claims] 1. An analytical method for measuring the concentration or activity of a specific substance in a liquid by detecting an optical change caused by a chemical reaction, wherein a plurality of optical concentrations or luminescence intensities are set at appropriate time intervals after the initiation of the reaction. By measuring the change in optical density by measuring twice, determine whether to continue the reaction from the value of optical density at each measurement point, and if it is determined that it is not necessary to continue the reaction from the viewpoint of analytical accuracy, When the calibration curve corresponding to the reaction time is selected and it is determined that the reaction needs to be continued, the reaction is further continued, and the analysis is completed in the minimum reaction time from the viewpoint of analysis accuracy. Analysis method.